Monoclonal antibody biosimilars for cancer treatment

Summary Monoclonal antibodies are important cancer medicines. The European Medicines Agency (EMA) approved 48 and the Food and Drug Administration (FDA) 56 anticancer monoclonal antibody-based therapies. Their high prices burden healthcare systems and hamper global drug access. Biosimilars could retain costs and expand the availability of monoclonal antibodies. In Europe, five rituximab biosimilars, six trastuzumab biosimilars, and eight bevacizumab biosimilars are available as anti-cancer drugs. To gain insight into the biosimilar landscape for cancer treatment, we performed a literature search and analysis. In this review, we summarize cancer monoclonal antibodies’ properties crucial for the desired pharmacology and point out sources of variability. The analytical assessment of all EMA-approved bevacizumab biosimilars is highlighted to illustrate this variability. The global landscape of investigational and approved biosimilars is mapped, and the challenges for access to cancer biosimilars are identified.


INTRODUCTION
Monoclonal antibodies are important cancer medicines.There are 48 approved by the European Medicines Agency (EMA) and 56 by the Food and Drug Administration (FDA), and these numbers will grow. 1Their global market burdens healthcare systems and hampers drug accessibility, with low-and middle-income countries having scarce to zero access. 2,3Biosimilars can potentially retain costs and expand drug availability. 4Monoclonal antibodies are complex macromolecules manufactured in living cells inherent to molecular heterogeneity, making it impossible to produce an exact copy.6][7] Unlike a chemically synthesized generic, approved upon a single bioequivalence study, biosimilar approval is based on ''the totality of evidence'' in a 3-layer similarity comparison with the originator. 8The upside-down triangle in Figure 1 emphasizes the analytical assessment in the first layer of biosimilar development, providing the most substantial evidence of similarity because even small differences are detected analytically.In the second layer, similarity of a biosimilar with the originator is assessed in preclinical models, although not required by EMA.The third and smallest layer consists of clinical studies, a phase 1 trial for pharmacokinetics and a phase 3 trial to confirm efficacy and safety for a sensitive indication in a homogenous population to detect the slightest differences with the originator. 8So far, EMA approved five anti-CD20 rituximab, seven anti-human epidermal growth factor receptor 2 (HER-2) trastuzumab, and eight anti-vascular endothelial growth factor (VEGF) bevacizumab anti-cancer biosimilars; FDA approved, respectively, three, five, and four. 9,10Despite the urgency, the availability and uptake of anti-cancer biosimilars are divergent among European countries. 11,12n this review, we outline current developments in the complex biosimilar field.In addition to existing reviews from a more regulatory perspective, 14,15 we first aim to provide background information on pharmacology of monoclonal antibody biosimilars for cancer, to increase understanding of potential variation and consequences of variation for safety, pharmacokinetics, and efficacy.To demonstrate the reliability of analytical assessments, we provide an overview of EMA-approved bevacizumab biosimilars as a case study.It demonstrates how elaborate and powerful the analytical assessment is, how low variation in practice is, and how the smallest variability is detected, that will not be picked up by large efficacy studies.Hereafter, we mapped available biosimilars worldwide and identified the challenges and opportunities for antibody biosimilar uptake in oncology.
biosimilars.From European Public Assessment Reports (EPARs), data regarding analytical assessment of the different approved bevacizumab biosimilars were summarized.Websites www.clinicialtrials.govand www.gabionline.netwere used to identify approved and investigational biosimilars for cancer indications.Additional biosimilars were found on PubMed, websites of pharmaceutical industries, and summaries of global market reports.The websites www.antibodysociety.org,and www.iqvia.comwere used to retrieve further relevant information regarding (biosimilar) monoclonal antibodies.Regulatory information was searched on www.who.int,www.ema.eu, and www.fda.gov.The definitions pertinent to this review are detailed in Table 1.

PHARMACOLOGY
Their complex structure and manufacture determine the pharmacology of monoclonal antibodies.Figure 2 shows monoclonal antibody manufacturing and sources of variability.Here, we summarize key components of their structural, functional, and product-related aspects and variability that could influence their pharmacological properties.After analytical characterization and non-clinical studies, originator approval relies on proof of clinical benefit vs. standard care.For biosimilars, the development phases' importance is opposite to the originator's: analytical assessment provides the strongest proof of similarity.In humans, a pharmacokinetic study has to be performed, and efficacy is evaluated in one main indication.EMA's guideline 13 on monoclonal antibody development describes parameters that need to be controlled regarding process, e.g., cell line stability, continuous capability to produce the desired product quality; impurities, viruses, function operational units, e.g., validation purification column, aseptic filling, column loads, pH, and temperature.Parameters regarding structure are identity, molecular weight, isoform pattern, extinction coefficient, electrophoretic profiles, chromatographic and spectroscopic profiles, antibody class, subclass, light-chain composition, primary structure e.g., peptide mapping, amino acid sequencing, and mass spectrometry analysis, N-and C-terminal amino acids e.g., C-terminal lysine(s), free sulfydryl groups, disulfide bridge integrity/mismatch, carbohydrate content, oligosaccharide pattern (neutral sugars, amino sugars, and sialic acids), N-glycosylation on heavy chains, other glycosylation site(s), glycan structures, mannosylation, galactosylation, fucosylation, sialylation, main glycan structure distribution (e.g., G0, G1, and G2).Regarding function, tests should reflect the clinic e.g., binding and neutralizing.Effector functions (also when not part of mechanism): ADCC, cytotoxic properties, complement binding and activation, C1q binding, Fc gamma-and neonatal receptor binding (cell-based assays preferred); antibody antigen affinity, avidity, and immunoreactivity; crossreactivity with immunohistochemistry; complementary determining regions; target epitope, e.g., protein, oligosaccharide, glycoprotein, glycolipid, amino acid sequence, and carbohydrate structure.Parameters regarding product are charge variants (quantitatively and qualitatively); chromatography/electrophoresis to detect truncation, dissociation, and polymerization, impurities: protein A, host cell proteins, DNA, culture or purification residues, downstream residues; C-terminal lysine processing, N-terminal pyroglutamate, deamidation, oxidation, isomerization, fragmentation, disulfide bond mismatch, N-linked oligosaccharide, and glycation (orthogonal methods).General tests involve drug quantity, appearance, solubility, pH, osmolality, extractable volume, sterility, bacterial endotoxins, and visible and subvisible particulate matter on batch release and for stability.

Structure
Figure 3 shows the typical Y-shape of the monoclonal antibody molecule, consisting of heavy and light chains subdivided into a variable and constant region.The variable region is antibody specific, whereas the constant region is most often the immunoglobulin backbone subtypes 1 or 4. 32,33 The variable fragment antigen-binding (Fab) region and the constant fragment crystallizable (Fc) region are responsible for functional properties, e.g., mechanism of action, effector function, and pharmacokinetics.The primary structure of a monoclonal antibody is the amino acid sequence, often humanized or fully human, but older constructs are chimeric (e.g., rituximab and cetuximab).Higher-order structures define the three-dimensional shape.The complementarity-determining region as part of the variable region enables antigen specificity.Disulfide bonds connect all regions for stability.The sugar groups at the constant heavy two domains of the Fc part, called the N-glycans, are important structures because of their strong influence on Fc-function.Novel antibodies are often glycoengineered at this site to influence half-life or effector functions. 34,35

Mechanism of action
Monoclonal antibodies can have different mechanisms of action.In short, monoclonal antibodies can directly target tumor cells by interfering with cell signaling or delivering a toxic payload such as antibody-drug conjugates. 32,36Another major class of monoclonal antibodies exerts immune-mediated tumor cell killing, such as immune checkpoint inhibitors, blocking the programmed death (ligand) 1 (PD-(L)1) axis. 37Immune-mediated tumor cell killing can also be caused by Fc-Fcg receptor interaction with macrophages or natural killer cells.This effect is

Analytical assessment:
Generation of quality, analytical, and functional data of a drug 5 Anti-drug antibody formation: An unwanted immune response against a therapeutic monoclonal antibody 16 Biological: A medicinal product whose active substance is made by or derived from a living organism 5 Biosimilar: The biological medicinal product, is highly similar to an already authorized biological medicinal product 5 Cost-effectiveness: Providing an extra year of healthy life for less than three times the Gross Domestic Product 17 Critical quality attribute: Physical, chemical, biological, or microbiological property or characteristic that should be within an appropriate limit, range, or distribution to ensure the desired product quality 18 Divergence: When a monoclonal antibody drifts or evolves 19 Drift: Unintended or unknown change in the manufacturing process of a monoclonal antibody 19 European public assessment report: A set of publicly available documents from EMA, with the complete developmental evaluation, product information, and medicine performance 20 Evolution: Deliberate changes in the manufacturing process for product improvement 19 Extrapolation of indication: The regulatory and scientific process of granting a clinical indication to a biosimilar extrapolated from one therapeutic indication, relying on the same mechanism of action, not requiring its own efficacy data 5,21 Generic: Chemically synthesized compounds with a simple, well-defined structure independent of the manufacturing process are easy to characterize completely 8

Immunogenicity:
The extent to which the host's immune system recognizes and reacts to a monoclonal antibody 22 Interchangeability: Refers to exchanging originators with their respective biosimilar, but also exchanging biosimilars that refer to the same originator product 8 Low-and middle-income countries: Economies with respectively $1,035 or less and between $1,036 and $12,535 gross national income per capita 23 Monoclonal antibody: An antibody derived from the clone of a single B cell produced in large quantities of identical cells possessing an affinity for the same epitope on a specific antigen, e.g., cancer cell 14 Originator: Innovative biological developed and patented by a pharmaceutical company 5 Pharmacology: Origin, chemistry, and uses of drugs and their effects on the body 24 Substitution: Automatically interchanging drugs at the pharmacy level 5 Switching: Interchanging originator and biosimilar or between biosimilars 5 WHO Essential Medicines List: Essential medicines that satisfy the priority healthcare needs of a population, selected for disease prevalence and public health relevance, evidence of efficacy and safety, and comparative cost-effectiveness.They are intended to be available in functioning health systems at all times 25 called antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). 36Lastly, monoclonal antibodies can influence the tumor microenvironment, such as vasculature, stroma, or soluble targets, e.g., when targeting VEGF with bevacizumab. 32,36

Pharmacokinetics
The pharmacokinetics of monoclonal antibodies is characterized by a fast distribution over large spaces, such as vasculature, due to their size and polarity, followed by slow elimination, with an elimination half-life of 11-30 days.Monoclonal antibodies have a unique target-mediated clearance via antigen binding, resulting in nonlinear pharmacokinetics at low doses.Most antibodies can be internalized after receptor binding and are then degraded via lysosomes, influenced by their dose, antigen affinity and density, and internalization rate.Non-specific monoclonal antibody clearance is the degradation in off-target cells, the liver, and the mononuclear phagocyte system via Fcg receptor binding.[40]

Safety
Apart from those related to their target, general safety concerns are infusion reactions and the extent of immunogenicity.The latter could lead to anti-drug antibody formation.This can result in reduced efficacy due to target neutralization and accelerated clearance.Human and humanized antibodies are better tolerated than chimeric or murine constructs due to the lower percentage of foreign parts. 41The degree of immunogenicity depends on drug dose, administration route, drug-related impurities, aggregates, and variable amino acid sequence or glycosylation and is influenced by the formulation. 16he aforementioned structural-, functional-, and product-related properties of monoclonal antibodies are so-called critical quality attributes.When changed, this variability could affect the mechanism of action, pharmacokinetics, and safety.This is summarized in Figure 3. Divided into upstream processing, involving gene transfection, stable cell clone selection, and antibody production from mammalian cells on small and large scales, and downstream processing, in which the antibody is recovered and purified through a combination of several methods.The product is then formulated, sterility filtrated, and packaged, followed by final release quality control (Created with biorender.com).LC, light chain; Fc, crystallizable fragment, Fd, heavy chain of the Fab region.With recombinant DNA technique, a vector with genes encoding for the variable and constant region is inserted into host cells, the ''expression system,'' that will produce the antibody for canonical antibodies, often Chinese hamster ovary or murine lymphoid cells.Expression systems have unique post-translational modifications: glycosylation, phosphorylation, deamidation, methylation, and acetylation, resulting in microheterogeneity, even between antibodies from the same cell line. 26Smaller constructs, lacking the highly variable Fc glycosylation, can be simpler produced in Escherichia coli bacteria. 27Immunoconjugates are more complex, with linker and toxin chemistry. 28,29Factors of influence are gene mutations in the host cell DNA, host cell impurities, cell productivity, and protein degradation, potentially leading to aggregates, fragments, unusual glycosylation forms, and charge-heterogeneity. Process parameters such as pH, pressure, temperature, and oxygen supply can also impact product quality. 30After antibody production in large bioreactors, isolation and purification steps remove cell-related impurities (host cell DNA, proteins), process-related impurities (buffers), and product-related impurities (aggregates and fragments).Finally, the monoclonal antibody is formulated, sterility filtrated, and packaged.Formulation buffers and storage conditions are critical for the protein's stability over time. 31

Variability
Manufactured monoclonal antibody batches, either originator or biosimilar, display inherently intermolecular heterogeneity, particularly in post-translational modifications, such as glycosylation.Moreover, manufacturing sites and processes are continuously subject to changes to improve production, increase production scale, or transfer to additional production sites. 22,42All these changes may affect quality attributes, potentially leading to changed pharmacology.Unwanted changes, so-called drifts, are rare, with only two reports for monoclonal antibodies used in oncology.One reported drift involved cetuximab, which inhibits epidermal growth factor receptor activation, first approved for colorectal cancer in 2004 in the European Union (EU) and the US in 2011.The cetuximab manufactured in the US showed a 22% higher drug exposure in patients due to decreased clearance than the EU-produced product. 43However, post-marketing comparison clinical trials revealed no difference in efficacy and safety. 44,45Another drift case involved the ADCC function of trastuzumab.ADCC is part of trastuzumab's mechanism of action. 46,47In several of the 203 trastuzumab originator batches, expiring between 2018 and 2019, two drifts were detected in the N-glycans' sugar residues.In the first case, decreased percentage of afucose caused decreased ADCC and FcgRIIIa binding.In a second drift case, an increased percentage of the high mannose sugar group caused increased ADCC activity and FcgRIIIa binding. 47A 3-year followup study revealed improved event-free survival of a biosimilar compared to the originator. 48Therefore, analysis of N-glycans' afucose and high mannose is crucial in the analytical assessment of trastuzumab originator and biosimilars. 499][50] The host cell and purification processes can influence the heterogeneity of monoclonal antibodies and thus potentially impact pharmacokinetics. 51Positively charged antibody variants are cleared faster nonspecifically than less positively charged variants (Figure 3). 38,40Analytical assessment continues during the drug's lifetime to verify that the monoclonal antibody remains similar. 52An extensive set of analytical methods evolved over three decades of protein manufacturing to detect the smallest variations. 22,53,54Therefore, the analytical assessment enables better distinction in potential differences than pharmacokinetics and efficacy studies in patients.For details, see Figure 1.

Review BEVACIZUMAB BIOSIMILARS
Each biosimilar application is carefully reviewed by EMA and approved based on a rigorous dataset, including human data for pharmacokinetics, safety, immunogenicity, and efficacy.6][57][58][59][60][61][62] During the comparative phase 1 and phase 3 clinical trials of the bevacizumab biosimilars, there were no differences and therefore no concerns, with regard to pharmacokinetics, safety, and efficacy.
Bevacizumab originator was approved by EMA in 2005 to treat patients with colorectal cancer and has been off-patent since 2022.With three additional biosimilars approved in 2022, bevacizumab counts with eight, the most biosimilars currently in Europe.For the details of the analytical assessment of all bevacizumab biosimilars, see Tables 2 and 3. EMA guidelines do not dictate specifically how and to what extent the analytical data should be provided, but the critical quality attributes that impact safety or efficacy should be extensively represented (see also Figure 1). 63The guidelines for ''biosimilar quality data'' are, in principle, based on the guideline for ''manufacturing changes for biological products.''Each biosimilar applicant should produce several batches of their product to be compared with the originator.A quality profile should be generated based on the analytical data of several clearly identified originator batches. 64,65The originator batches' variability range determines the biosimilar batches' specification limits.Quantitative ranges should be established where possible.When parameters are out of range, this should be accompanied by a justification for why this will not impact product quality, safety, or efficacy. 63EPARs summarize the total data collected in a redacted format.Although EPARs are structured similarly, they vary because they are the result of varying assessors and negotiation between EMA and the sponsor.Therefore, not only the methods and extent of analysis but also the publicly available data and the way of reporting vary from biosimilar to biosimilar, as is also demonstrated in our overview presented in Table 2.
We found that multiple parameters were evaluated for each attribute, reporting several tests per parameter, ranging from 18 to 52 tests per biosimilar.In Table 2, we summarized detected differences for each biosimilar.MB02 and CT-P16 showed minor differences at the N-and C terminal and different amounts of free thiol compared to the originator.For each biosimilar, differences in sugar residues are detected.Increased high mannose for MYL-1402O was out of range.However, this was accepted since no clinical impact was revealed in the registration data.The apparent different relative binding affinity to VEGF of PF-06439535 compared to the originator, an important aspect of bevacizumab's mode of action, was within the quantitative range.Related to glycan group variability, each biosimilar also poses differences in the Fc effector functions.Several bevacizumab biosimilars have a reduced amount of high-molecular-weight species, leading to a better safety profile than the originator.Each biosimilar also shows variability in acidic-, basic-, or main charge species.However, the reported differences have no clinical impact.For instance, the slightly higher mannose glycan count of ABP215 did not increase serum half-life in patients. 59In general, the overview shows a difference in the number of tests.This could be explained by a distinction between critical quality attributes and quality attributes.Variability of the N terminus, for example, is a quality attribute, mandatory to characterize a biosimilar candidate.However, it is not a critical quality attribute, which allows for more loose variability margins.In contrast, the amino acid sequence, a critical quality attribute, needs to be identical.Glycosylation variability could impact safety, pharmacokinetics, and efficacy.It is therefore defined as a critical quality attribute and thoroughly tested.This also applies to all the functional properties, on both the Fc (ADCC and CDC) and the Fab region (antigen-binding).Fc effector function is responsible for ADCC, and CDC functions, and could potentially be affected by variability in glycosylation sites.Therefore, glycosylation as well as ADCC and CDC function undergo in-depth analysis, regardless of their role in the mechanism of action.This is demonstrated in our overview for bevacizumab, a drug that lacks ADCC and CDC function.The absence of the ADCC and CDC function is proven in cell-based assays for each new biosimilar (Tables 2 and 3).Moreover, there is a distinction between orthogonal testing providing multiple answers (mass spectrometry, for example, gives information on peptide mapping and molecular weight) and linear testing providing single answers (e.g., cell-binding assays). 66,67In summary, most data are provided for Fc-function parameters, namely >20% of total data, displaying 80% similarity with the originator.The least data are provided for charge variants, <10%, showing the highest variability with 50% similarity.The highest levels of similarity were observed in primary structure (90%), higher-order structure (91%), and Fab function (96%), in line with the required identical amino acid sequence and mode of action.Glycosylation parameters were 58% similar, and data regarding molecular weight and impurities presented 54% similarity.These calculations do not include function-and product-related parameters of CT-P16 due to a lack of provided information in the EPARs.In conclusion, the data in the EPARs of bevacizumab biosimilars demonstrate a rigorous registration dataset on all clinically relevant attributes.

LANDSCAPE
Now that it is clear that originators and their biosimilars are clinical equivalents, grounded mainly on analytical data, next, we pursued to map a global biosimilar landscape (Figure 4).The landscape provides an impression of biosimilars under preclinical and clinical evaluation and approved biosimilars, not only by EMA but also outside Europe.This overview is meant as an inventory of what is currently available and what may be expected until 2028.Interestingly, for some monoclonal antibodies, their market exclusivity has expired.Although not all licensed by advanced agencies yet, a few biosimilars have become available, namely for ado-trastuzumab emtansine, cetuximab, panitumumab, brentuximab-vedotin, and ipilimumab.In the EU, even fewer are available, namely for only 3 out of 8 monoclonal antibodies for cancer, with expired patents. 68The high amount of biosimilars for trastuzumab and bevacizumab matches the large patient populations that can be treated with monoclonal antibodies and therefore, market size. 69The rituximab biosimilar proportion is smaller but significant with indications for autoimmune diseases such as rheumatoid arthritis and included even on World Health Organization's (WHO) Essential Medicines List for diffuse large B cell lymphoma, chronic lymphocytic leukemia, and follicular lymphoma. 25India and China are prominently represented in biosimilar development; however, be aware of the fact that not all countries in this overview have national regulatory authorities with equal WHO     [56][57][58][59][60][61][62][63] maturity levels (Table 4).Interesting phenomena are the PD-1 and PD-L1-targeting monoclonal antibodies that continuously enter the market. 1Although labeled innovative, they are rather ''me-too'' drugs, but not biosimilars.Despite distinct structural differences, they are considered interchangeable. 70For instance, pembrolizumab is a humanized antibody, binding with a different affinity to a different PD-1 epitope than the fully human antibody nivolumab, meaning that their structure, especially their complementary determining regions are entirely different, as well as the amino acid sequence of both compounds. 71However, they show highly similar clinical efficacy. 70Given the upcoming EU patent expirations of nivolumab (2026) and pembrolizumab (2028), there are likely more biosimilars within invisible pipelines than we could find.The development and use of PD-1 antibody biosimilars will have a major clinical and financial impact, considering that nivolumab and pembrolizumab are dominating the global antibody market. 69Rituximab, trastuzumab, nivolumab, and pembrolizumab (the last two only for metastatic melanoma) are on WHO's Essential Medicines List, which makes it paramount that they become globally available and affordable as biosimilars.

CHALLENGES
The themes identified as challenges for cancer monoclonal antibody biosimilars extracted from our search were development costs and drug prices, confirmatory efficacy trials, patents, access in low-and middle-income countries, implementation, and interchangeability.4 for details and reference per drug.No biosimilars were found for CD20-targeting ofatumumab and obinutuzumab (off-patent 2023 and 2024), EGFR-targeting panitumumab (off-patent 2018), VEGFR2-targeting ramucirumab (off-patent 2023), or CD30-targeting brentuximab vedotin (off-patent 2021).CD, cluster of differentiation; CTLA-4, cytotoxic T lymphocyte-associated protein 4; EGFR, epidermal growth factor receptor; EMA, European Medicines Agency; HER-2, human epidermal growth factor receptor 2; PD-1, programmed death 1; VEGF(R2), vascular endothelial growth factor (receptor 2).Be aware of the fact that not all countries included in this overview have the same maturity level, based on WHO standards on national regulatory authorities.ADC, antibody-drug conjugate; CD, cluster of differentiation; CTLA-4, cytotoxic T lymphocyte-associated protein 4; EGFR, epidermal growth factor receptor; EMA, European Medicines Agency; HER-2, human epidermal growth factor receptor 2; PD-1, programmed death 1; VEGF(R2), vascular endothelial growth factor (receptor 2).Be aware of the fact that not all countries included in this overview have the same maturity level, based on WHO standards on national regulatory authorities.Note: Countries indicated with an * have a maturity level 3 with regard to their regulatory system, according to WHO standards (stable, well-functioning, and integrated).The other countries have a maturity level 4 (Advanced level of performance and continuous improvement). 109

Development costs and drug prices
The first hurdle in developing monoclonal antibody biosimilars is their complex and therefore costly manufacturing and extensive analytical assessment. 110Development can be improved to some extent by computational tools, in silico methods, and innovative high-throughput technologies. 111Strikingly, between now and 2030 for less than 50% of exclusivity-expiring molecules, biosimilars are in the pipeline. 12It seems that even in rich countries, at this moment, the investment to develop a biosimilar might not be attractive among other reasons due to high demands by regulators. 112The majority of development costs are spent on clinical studies.Reducing the mandatory clinical data as currently being explored by regulators might improve prospects. 113,114Also, improvements in analytical testing and modeling alternatives could provide smart solutions and might further reduce costs. 14There is great variety in cost-effectiveness among countries, and not a straightforward answer to cost-effectiveness of biosimilars.Apart from the lack of transparency in development costs, this is also due to the differences in healthcare systems.It is therefore difficult to compare different countries.Factors that play a role are for example local, regional, and national health policy; local and central government decisions on reimbursement; and distribution of drugs to hospitals. 15hereas generics can be sold for a fraction of the original drug price, cost savings of biosimilars is divergent in Europe.The modest list price reductions so far show that most robust savings should be in Poland and Germany, with 46% and 40% price reduction, whereas in the United Kingdom and Norway, prices increased with 10% and 5%. 12,115Following savings on antibodies in the rheumatology therapeutic area, we may expect up to 69% price reductions due to biosimilar use. 116,117Several studies predicted favorable cost-effectiveness of monoclonal antibody biosimilars in cancer.9][120][121][122][123] At this moment, for the industry, the low biosimilar prices in the competitive market do not warrant biosimilar development at relatively high costs.

Confirmatory efficacy trials
5][126] In some countries, such as Sweden and the US, additional data on switching from originator to biosimilars are required to allow interchangeability. 127,128Two systemic reviews on biosimilar switching studies, including cancer monoclonal antibodies, show no clinically meaningful differences after switching. 129,130In addition, evidence is building that phase 3 trials might not add additional value for biosimilars. 131,132This creates a discussion in health organizations to adjust the approval pathway.For example, the Medicines and Healthcare Products Regulatory Agency in the United Kingdom stated in May 2021 that confirmatory efficacy trials would no longer be necessary for biosimilars when scientifically justified. 133EMA-associated scientists analyzed all approved biosimilars and concluded that in none of the approvals the patient trial played a decisive role. 134Recently, the EMA published a summary of analytical data of bevacizumab and adalimumab biosimilars.They confirm our findings regarding our analysis of the EPARs of the bevacizumab biosimilars.They firmly stated that clinical efficacy data were of low relevance regarding quality concerns, urging to redefine the requirements of the clinical evaluation of biosimilars. 21In addition, they investigate the suitability of biosimilar development based on quality data, and waiver of clinical efficacy and safety trials on a case-by-case approach reported in their scientific advice from September 2022. 135This may result in faster and cheaper availability of biosimilars.The development time for an innovative monoclonal antibody is generally 10-12 years, and for a biosimilar still 8-10 years 136 During the COVID-19 pandemic, the launch of anti-COVID-19 antibodies happened within several months.The licensing of the product and patent suspension were being discussed by governments. 137,138The same collaboration could support and broaden the availability of cancer monoclonal antibodies and their biosimilars.[141]

Patents
Originator patent expirations allow for healthy market competition with their biosimilars, which is expected to decrease the global costs of all EMA-approved biosimilars for the 3 reference products, namely rituximab, trastuzumab, and bevacizumab for the targets CD20, HER-2, and VEGF, respectively. 69The exponential growth of the monoclonal antibody market over the last 10 years, especially for cancer indications, partially explains the current gap between the high number of marketed originators and the low number of marketed biosimilars.It also indicates that a similar exponential biosimilar market growth might be expected once market exclusivities start lifting further.However, patents for these complex products are long, generally 10 years in the EU and 12 years in the US, with potential extensions. 142This time allows the innovator company to cover the high development costs and make profits and drives innovation.Development costs of new medicines are for the major part capital costs, and there is no relation with drug prices and manufacturing costs.However, the current development costs for an innovator company is not in line with what the system pays for a new drug, respectively 200-300 million vs. 2-3 billion US dollars as reported by Strategies in Regulated Markets. 113In the EU patent, regulations are overall liberal and different per country.This allows biosimilar developers to wait out key patents in most countries or launch a biosimilar at risk, with no or low punitive damage, in, for instance, the Netherlands. 143ong patents are an issue in the US, more than in other countries.Many patents can be applied for, also after market launch, in addition to product and manufacture. 144To avoid infringements in the US between originator and biosimilar companies, a so-called ''patent dance'' is put in place, involving back-and-forth communication between the competing companies. 145Pembrolizumab exemplifies an over-patented drug in the US.Of more than 100 patent applications, 53 have been granted.Pembrolizumab's patent duration is extended with 8 years, with estimated extra drug costs of $137 billion. 146Pembrolizumab is expected to predominate the monoclonal antibody market by 2024, with a predicted $18 billion global annual sales. 147Patent strategies by pharmaceutical companies to keep a product exclusive are the addition of new indications (e.g., glioblastoma for bevacizumab), a new route of administration (e.g., subcutaneous for trastuzumab), or new drug formulations. 148Another opportunity for biosimilar companies is a ''skinny label,'' which refers to the approval pursuit of a biosimilar for a single indication and not all indications for which the brand name of the drug is approved.Skinny labeling is a measure to create early competition of biosimilars with their originators, paramount, since this will likely lead to substantial cost savings. 149Medicines Patent Pool is an initiative, striving through voluntary licensing and patent pooling, to allow valuable medicines to reach low-and middle-income countries. 150cess in low-and middle-income countries Biosimilars are an entrance to cancer treatment for countries lacking the resources for innovative monoclonal antibodies.There is a communication and knowledge barrier between low-and middle-income countries and supporting agencies, e.g., WHO, that needs to be bridged. 149n order to reach low-and middle-income countries, key elements were recently identified, namely, prioritizing targets according to impact on public health, supporting biosimilar development, market-entry, and use, in a country-specific manner. 151An overview of biosimilar access in 40 countries based on licensing is given by Huang et al. 152 Their review showed that Asia has the most biosimilars available, whereas, for Africa, there was only one biosimilar for rituximab at that time.They stated, too, that determining the actual access is far more complex, depending on barriers such as government reimbursement, out-of-pocket costs, budget allocations for biosimilars, shortages, and patent rights. 153,154The authors of these papers state that there is no simple solution to balancing universal guidelines and country-specific needs.Another article describes legal and regulatory issues in such countries, lack of research infrastructure, and educational barriers. 155The availability of rituximab biosimilars in India has dramatically improved treatment access, from 35% to 95% of the patients with large B cell lymphoma. 156Furthermore, a comparison study between US and India reveals that the treatment of these patients is now similar between these two countries. 157Several papers describe the concerns of biosimilar use in Latin America, such as non-adherence to already inconsistent regulations.There is also a need for traceability and pharmacovigilance of biosimilars and precise use of interchangeability.This requires educational efforts in Latin America. 1580][161] A budget impact analysis in 13 countries in the Middle East and North Africa predicted a substantial cost-saving effect of a rituximab biosimilar, assuming a 30% lower drug price. 162This indicates the importance of biosimilars in becoming more available in their markets.Several goals are pursued by health organizations such as the American Society of Clinical Oncology (ASCO), European Society of Medical Oncology (ESMO), and WHO, e.g., increasing global access to WHO's Essential Medicines List, working toward establishing value-based drug pricing, and pricing based on country-specific recourses and cancer burden. 163,164Moreover, between 2019 and 2022, 6 rituximab products and 10 trastuzumab products have been approved via the WHO prequalification program. 165[168][169][170]

Implementation and interchangeability
Biosimilar market penetration requires active promotion and financial awareness of stakeholders, e.g., payers, pharmacists, prescribers, and patients. 171In Europe, Denmark and the Netherlands switch the most whereas Bulgaria and Belgium performed worst. 12Recently, a policy review concluded how EU, US, and Japanese regulations could be improved by addressing region-specific competition barriers and educational needs. 172Health organizations, e.g., ASCO, ESMO, and WHO, have already undertaken initiatives to provide biosimilar information and education for healthcare providers and patients. 155,163,173,1749][180][181][182][183] EMA has now released the statement that biosimilars are, upon approval, interchangeable with their originator and with biosimilars referring to the same reference product.This will increase their use. 30This will eventually also allow the extrapolation of indication, using biosimilars for off-label indications relying on the proven mechanism of action they have been approved for.There is, however, still a conceptual difference of the meaning of interchangeability as used in the US compared to the rest of the world.In the US, interchangeability is a specific legal status for a biosimilar, awarded by the FDA upon fulfilling considerable additional requirements, such as a multi-switch trial.This may confuse prescribers in the world, suggesting 2 standards for biosimilars. 184

CONCLUDING REMARKS
The current landscape of biosimilars to treat cancer indicates that at the moment, biosimilar development may not be an attractive investment.To fully exploit biosimilar development and use, slimming of the clinical data package might be essential as is already explored by regulators.Much biosimilar development is going on, yet their increased uptake and cost-saving effect can only happen if challenges, described in our review, are tackled.Opportunities for improvement in this highly complex field lay in the pricing, reimbursement, and long patent duration for originator monoclonal antibodies.These issues are being addressed by close interaction between regulators, health technology assessment bodies, and other relevant initiatives, such as Medicines Patent Pool.Lastly, a continuous provision of knowledge and financial awareness is paramount, pursued by ASCO, ESMO, and WHO, which will eventually lead to affordable monoclonal antibody cancer treatments and access to it in all corners of the world.

Figure 1 .
Figure 1.Development phases biosimilar versus originatorAfter analytical characterization and non-clinical studies, originator approval relies on proof of clinical benefit vs. standard care.For biosimilars, the development phases' importance is opposite to the originator's: analytical assessment provides the strongest proof of similarity.In humans, a pharmacokinetic study has to be performed, and efficacy is evaluated in one main indication.EMA's guideline13 on monoclonal antibody development describes parameters that need to be controlled regarding process, e.g., cell line stability, continuous capability to produce the desired product quality; impurities, viruses, function operational units, e.g., validation purification column, aseptic filling, column loads, pH, and temperature.Parameters regarding structure are identity, molecular weight, isoform pattern, extinction coefficient, electrophoretic profiles, chromatographic and spectroscopic profiles, antibody class, subclass, light-chain composition, primary structure e.g., peptide mapping, amino acid sequencing, and mass spectrometry analysis, N-and C-terminal amino acids e.g., C-terminal lysine(s), free sulfydryl groups, disulfide bridge integrity/mismatch, carbohydrate content, oligosaccharide pattern (neutral sugars, amino sugars, and sialic acids), N-glycosylation on heavy chains, other glycosylation site(s), glycan structures, mannosylation, galactosylation, fucosylation, sialylation, main glycan structure distribution (e.g., G0, G1, and G2).Regarding function, tests should reflect the clinic e.g., binding and neutralizing.Effector functions (also when not part of mechanism): ADCC, cytotoxic properties, complement binding and activation, C1q binding, Fc gamma-and neonatal receptor binding (cell-based assays preferred); antibody antigen affinity, avidity, and immunoreactivity; crossreactivity with immunohistochemistry; complementary determining regions; target epitope, e.g., protein, oligosaccharide, glycoprotein, glycolipid, amino acid sequence, and carbohydrate structure.Parameters regarding product are charge variants (quantitatively and qualitatively); chromatography/electrophoresis to detect truncation, dissociation, and polymerization, impurities: protein A, host cell proteins, DNA, culture or purification residues, downstream residues; C-terminal lysine processing, N-terminal pyroglutamate, deamidation, oxidation, isomerization, fragmentation, disulfide bond mismatch, N-linked oligosaccharide, and glycation (orthogonal methods).General tests involve drug quantity, appearance, solubility, pH, osmolality, extractable volume, sterility, bacterial endotoxins, and visible and subvisible particulate matter on batch release and for stability.

Figure 2 .
Figure 2. Monoclonal antibody manufacturingDivided into upstream processing, involving gene transfection, stable cell clone selection, and antibody production from mammalian cells on small and large scales, and downstream processing, in which the antibody is recovered and purified through a combination of several methods.The product is then formulated, sterility filtrated, and packaged, followed by final release quality control (Created with biorender.com).LC, light chain; Fc, crystallizable fragment, Fd, heavy chain of the Fab region.With recombinant DNA technique, a vector with genes encoding for the variable and constant region is inserted into host cells, the ''expression system,'' that will produce the antibody for canonical antibodies, often Chinese hamster ovary or murine lymphoid cells.Expression systems have unique post-translational modifications: glycosylation, phosphorylation, deamidation, methylation, and acetylation, resulting in microheterogeneity, even between antibodies from the same cell line.26Smaller constructs, lacking the highly variable Fc glycosylation, can be simpler produced in Escherichia coli bacteria.27Immunoconjugates are more complex, with linker and toxin chemistry.28,29Factors of influence are gene mutations in the host cell DNA, host cell impurities, cell productivity, and protein degradation, potentially leading to aggregates, fragments, unusual glycosylation forms, and charge-heterogeneity. Process parameters such as pH, pressure, temperature, and oxygen supply can also impact product quality.30After antibody production in large bioreactors, isolation and purification steps remove cell-related impurities (host cell DNA, proteins), process-related impurities (buffers), and product-related impurities (aggregates and fragments).Finally, the monoclonal antibody is formulated, sterility filtrated, and packaged.Formulation buffers and storage conditions are critical for the protein's stability over time.31

Figure 4 .
Figure 4. Monoclonal antibody biosimilars in cancer Biosimilars (EMA-)approved, and in (pre-)clinical phase found for cancer indications, patent phase in the European Union indicated per drug.When the same drug is licensed under two trade names, this number is shown in brackets.See Table4for details and reference per drug.No biosimilars were found for CD20-targeting ofatumumab and obinutuzumab (off-patent 2023 and 2024), EGFR-targeting panitumumab (off-patent 2018), VEGFR2-targeting ramucirumab (off-patent 2023), or CD30-targeting brentuximab vedotin (off-patent 2021).CD, cluster of differentiation; CTLA-4, cytotoxic T lymphocyte-associated protein 4; EGFR, epidermal growth factor receptor; EMA, European Medicines Agency; HER-2, human epidermal growth factor receptor 2; PD-1, programmed death 1; VEGF(R2), vascular endothelial growth factor (receptor 2).Be aware of the fact that not all countries included in this overview have the same maturity level, based on WHO standards on national regulatory authorities.

Table 3 .
Detailed version of Table 2: analytical assessments of bevacizumab biosimilars

Table 4 .
Monoclonal antibody biosimilars in cancer: details of Figure4